U.S. patent application number 14/111647 was filed with the patent office on 2014-02-06 for novel paenibacillus sp. hpl-001 strain for producing highly active xylanase having heat resistance and a wide ph range, novel xylanase enzyme isolated therefrom, and method for mass-producing same using a transformant thereof.
This patent application is currently assigned to KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY. The applicant listed for this patent is In Taek Hwang, He Kyoung Lim, No Joong Park. Invention is credited to In Taek Hwang, He Kyoung Lim, No Joong Park.
Application Number | 20140038262 14/111647 |
Document ID | / |
Family ID | 47715222 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038262 |
Kind Code |
A1 |
Hwang; In Taek ; et
al. |
February 6, 2014 |
NOVEL PAENIBACILLUS SP. HPL-001 STRAIN FOR PRODUCING HIGHLY ACTIVE
XYLANASE HAVING HEAT RESISTANCE AND A WIDE PH RANGE, NOVEL XYLANASE
ENZYME ISOLATED THEREFROM, AND METHOD FOR MASS-PRODUCING SAME USING
A TRANSFORMANT THEREOF
Abstract
The present invention relates to the novel Paenibacillus sp.
strain, and the novel protein isolated from the same. More
particularly, the present invention relates to the novel
Paenibacillus sp. strain producing xylanase, and the novel xylanase
having high activity at high temperature and in a wide range of pH,
and a production method of the same. The Paenibacillus sp. HPL-3
strain (KCTC11987BP) and the xylanase of the present invention
demonstrates high activity at high temperature or in a wide range
of pH to decompose xylan, the major component of various
lignocellulosic biomass, so that they can be effectively used for
the production or development of bio-fuel, alternative material,
performance chemical, bio-polymer, food and feeds, etc.
Inventors: |
Hwang; In Taek; (Daejeon,
KR) ; Park; No Joong; (Daejeon, KR) ; Lim; He
Kyoung; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hwang; In Taek
Park; No Joong
Lim; He Kyoung |
Daejeon
Daejeon
Daejeon |
|
KR
KR
KR |
|
|
Assignee: |
KOREA RESEARCH INSTITUTE OF
CHEMICAL TECHNOLOGY
Taejeon-si
KR
|
Family ID: |
47715222 |
Appl. No.: |
14/111647 |
Filed: |
August 12, 2011 |
PCT Filed: |
August 12, 2011 |
PCT NO: |
PCT/KR2011/005966 |
371 Date: |
October 14, 2013 |
Current U.S.
Class: |
435/209 ;
435/252.33; 435/254.2; 435/325; 435/348 |
Current CPC
Class: |
C12Y 302/01008 20130101;
C12N 9/248 20130101; A23K 50/75 20160501; C12N 9/2482 20130101;
A23K 20/189 20160501; A23K 50/10 20160501 |
Class at
Publication: |
435/209 ;
435/252.33; 435/254.2; 435/325; 435/348 |
International
Class: |
C12N 9/24 20060101
C12N009/24 |
Claims
1. A Xylanase having a) the amino acid sequence represented by SEQ.
ID. NO: 4; b) the amino acid sequence having at least 70% homology
with the amino acid sequence represented by SEQ. ID. NO: 4; c) the
amino acid sequence encoded by the nucleotide sequence represented
by SEQ. ID. NO: 3; d) the amino acid sequence of the protein
composed of the modified amino acid sequence having substitution,
deletion, insertion, and/or addition of at least one of amino acids
of the sequence represented by SEQ. ID. NO: 4, which is also
identical in its function to the protein containing the amino acid
sequence represented by SEQ. ID. NO: 4; or e) the amino acid
sequence encoded by DNA hybridized with the DNA containing the
nucleotide sequence represented by SEQ. ID. NO: 3 in the strict
condition, which is identical in its function to the protein
containing the amino acid sequence represented by SEQ. ID. NO:
4.
2. The xylanase according to claim 1, wherein the xylanase having
the characteristics of (a).about.(d): (a) molecular weight:
approximately 65 KDa on SDS-PAGE; (b) maximum activity in the range
of pH 5.about.pH 11; (c) maximum activity at the temperature of
50.about.60.degree. C.; and, (d) xylose production at 50.degree.
C., pH 5.about.pH 11: at least 95 unit/min mg; and xylose
production at 60.degree. C., pH 6.about.pH 9: at least 100 unit/min
mg.
3-7. (canceled)
8. A transformant having host cells to which a recombinant
expression vector is introduced, in which polynucleotide encoding
the xylanase of claim 1 is operably linked to the recombinant
expression vector.
9. The transformant according to claim 8, wherein the host cells
are prokaryotic cells including E. coli or eukaryotic cells
including yeast, animal cells and insect cells.
10. The transformant according to claim 8, wherein the
polynucleotide has one of the following sequences: a) the
nucleotide sequence represented by SEQ. ID. NO: 3; b) the
nucleotide sequence having 90% homology with the nucleotide
sequence represented by SEQ. ID. NO: 3; c) the nucleotide sequence
encoding the amino acid sequence represented by SEQ. ID. NO: 4; d)
the nucleotide sequence encoding the amino acid sequence of the
protein with substitution, deletion, insertion, and/or addition of
at least one of amino acids of the sequence represented by SEQ. ID.
NO: 4, which is also identical in its function to the protein
containing the amino acid sequence represented by SEQ. ID. NO: 4;
and, e) the nucleotide sequence comprising DNA sequence to be
hybridized with DNA containing the nucleotide sequence represented
by SEQ. ID. NO: 3 under the strict condition and encoding the
protein having the same function as the protein comprising the
amino acid sequence represented by SEQ. ID. NO: 4.
11. The production method of xylanase comprising the steps of: a)
obtaining crude enzyme solution by centrifugation after culturing
Paenibacillus sp. HPL-3 strain or the strain of claim 3 in a
medium; and b) purifying xylanase from the crude enzyme solution of
step a).
12. The production method according to claim 11, wherein the strain
is Paenibacillus sp. HPL-3 deposited under the Accession No. of
KCTC11987BP.
13. The production method according to claim 11, wherein the
xylanase has one of the following sequences: a) the amino acid
sequence represented by SEQ. ID. NO: 3; b) the amino acid sequence
having 95% homology with the amino acid sequence represented by
SEQ. ID. NO: 3; c) the amino acid sequence encoded by the
nucleotide sequence represented by SEQ. ID. NO: 4; d) the amino
acid sequence of the protein composed of the modified amino acid
sequence having substitution, deletion, insertion, and/or addition
of at least one of amino acids of the sequence represented by SEQ.
ID. NO: 4, which is also identical in its function to the protein
containing the amino acid sequence represented by SEQ. ID. NO: 4;
and e) the amino acid sequence encoded by DNA hybridized with the
DNA containing the nucleotide sequence represented by SEQ. ID. NO:
3 in the strict condition, which is identical in its function to
the protein containing the amino acid sequence represented by SEQ.
ID. NO: 4.
14-22. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel Paenibacillus sp.
strain producing novel xylanase, a novel xylanase separated from
the strain, and a method for mass-production of the same using the
transformant of the strain. More precisely, the said novel xylanase
exhibits excellent activity of decomposing xylan at high
temperature or in a wide range of pH, so that the novel enzyme can
be effectively used not only in the fields of feeds, paper and
detergent industries, but also in the field of biochemical industry
producing bio-fuel, petroleum alternative fuel, performance
chemical, or bio-polymer.
[0003] 2. Description of the Related Art
[0004] To keep pace with the changes of international environmental
regulations including reduction of carbon dioxide emission,
humankind of 21.sup.st century has the assignment to develop
alternative resources of fossil fuel. In preparation of global
warming and oil resource depletion, solar energy, wind power,
hydropower, atomic power, and biomass have been major targets of
research. In the process of biorefinery for the production of
bio-fuel and chemical fuel via saccharification of lignocellulosic
biomass, it is inevitable to produce the by-product, xylan, that is
included in the lignocellulosic biomass by 15-30%. However, it is
not possible to use the by-product directly, so most of it are
discarded as wastes.
[0005] Hydrolysis of xylan has been induced via chemical method so
far, which is precisely as follows: sulfuric acid is added to
lignocellulosic biomass, followed by decomposing at 130.degree. C.
with pressurized steam; leading to the conversion into xylose and
xyloologosaccharide. However, in the above process, many impurities
are additionally generated by excessive reaction. Therefore,
purifying technique is additionally requested. This method has
other disadvantages as follows: it not only consumes a massive
amount of energy but also requires high priced equipments durable
in acid and high temperature condition, and it costs extra money
for the treatment of wastes generated during the processes, raising
the production cost as well. On the other hand, the biological
method for xylan decomposition using xylanase (the enzyme that
converts xylan, the major component of hemicellulose, into xylose
via saccharification is generally called xylanase) consumes less
energy and produces less wastes, compared with the chemical method,
suggesting that it has an economical advantage owing to the easy
treatment of less wastes.
[0006] Xylanase is not only urgently requested in the process of
biorefinery (biochemical industry) but also utilized in the process
of paper bleaching, for the improvement of feeds efficiency, in the
clearing process of fruit beverages, for the production of high
quality bread, and in the utilization of agricultural by-products,
etc. Xylanase has been produced by using various microorganisms up
to date. In particular, alkali-resistant or heat-resistant xylanase
was isolated from various bacteria for the use in breaching process
of paper industry (Tenkanen, M. et. al., Enzyme. Microb. Technol,
14, 566-574, 1992). Many xylanase producing microorganisms without
cellulase activity have been reported, and attempts to reduce
cellulose loss in the production of paper have been also reported
(Khashin, A. et. al., Appl. Environ. Microbiol, 59, 1725-1730,
1993; Kosugi, A. et. al., J. Bacteriol, 183, 7037-7043, 2001).
Successful cases have been reported to improve quality of bread by
treating xylanase (Courtin, C. M. et. al., J. Agric. Food. Chem.,
47, 1870-1877, 1999), and to introduce .beta.-xylosidase and
xylanase genes into yeast that is further used for saccharification
of agricultural by-products for a microorganism to use them (La
Grange, D. C. et. al., Appl. Environ. Microbiol, 67, 5512-5519,
2001). In the feeds industry, xylanase came into the market as a
feed additive enzyme. So, when the cattle eat grain feeds
containing the feed additive enzyme, the enzyme is functioning
lower viscosity generated by hemicellulose of the intestines of the
cattle, indicating that it is helpful to prevent digestive disease
in the cattle and to improve feed efficiency (McCracken, K. J. et.
al., Br. Poult. Sci, 42, 638-642, 2001).
[0007] Among the xylanase producing microorganisms reported so far,
Trichoderma sp. fungal strains have been largely used whose enzyme
productivity is superior than other xylanase producing bacteria but
the maximum activity is mainly observed in acidic condition
(Tenkanen at al., Enzyme and Microbial Technology 14(7):566-574,
1992). Xylanase producing bacteria are exemplified by Aeromonas
sp., Bacillus sp., Clostridium sp., Streptomyces sp., Aspergillus
sp, etc. The properties of xylanase depend on the bacteria and
various genes able to encode xylanase have also been reported.
[0008] Status of domestic and international technology involved in
xylanase is as follows. Trichoderma sp. C-4 strain producing
cellulase was identified by Dr. Jung's research team of Kyung Hee
University, Korea, however, higher activity is required for the
industrialization (Sul et al., Appl Microbiol Biotechnol.
66(1):63-70, 2004). Cephalosporium sp. RYM-202 strain producing
alkali-resistant xylanase was identified by Dr. Kang's research
team of Donghae University, Korea, and its usability in pulp
processing is being studied (Kang et al., Korean Journal of
Environmental Biology 17(2):191-198, 1999). Bacillus subtilis
DB104/pJHKJ4, the recombinant strain producing Bacillus originated
endoxylanase, was constructed by Dr. Kim's research team of KAIST,
Korea (Kim J H et al., J. Microbiol. Biotechnol., 10(4):551-553,
2000). In Taiwan, a case has been reported that alkali-resistance
of xylanase genetically replicated from the anaerobic fungus
Neocallimastix patriciarum was increased by directed enzyme
evolution (Yew-Loom Chen et al., Can. J. Microbiol.
47(12):10881094, 2001) and recently Bacillus firmus, one of the
alkali-resistant strains, has been identified in waste water
generated from the pulp processing (Pochih Chang, Biochemical and
Biophysical Research Communications 319:1017-1025, 2004). This
xylanase demonstrates high activity in the wide pH range of
4.about.11 and heat-resistance as high as maintaining 70% of the
original activity even after 16 hour culture at 62.degree. C.
Likewise, various strains have been developed and their functions
have been improved via directed evolution in many countries.
[0009] Patents in relation to xylanase so far are mainly focused on
the method for producing xylanase by using a recombinant strain
obtained from E. coli or using wild-type strain identified as
xylanase producing one (International Patent Publication No.
93/08275, International Patent Publication No. 92/01793,
International Patent Publication No. 92/17573, Korean Patent
Publication No. 10-0072225, Korean Patent Publication No.
10-02211204, Korean Patent Publication No. 10-0411771). Patents in
relation to xylanase as a feed additive so far are as follows:
Novel Streptomyces sp. WL-2 strain producing xylanase (Korean
Patent Publication No. 2001-0111986); Recombinant plasmid
containing secretion signal sequence of endoxylanase from Bacillus
subtilis and expression of foreign proteins using thereof (Korean
Patent Publication No. 2000-0034279); and Gene coding xylanase of
Bacillus sp. AMX-4 strain and recombinant xylanase through
transformant thereof (Korean Patent Publication No.
2003-0085679).
[0010] Domestic patents in relation to xylanase so far are as
follows: Novel Streptomyces sp. WL-2 strain producing xylanase
(Korean Patent Publication No. 2001-0111986); Recombinant plasmid
containing secretion signal sequence of endoxylanase from Bacillus
subtilis and expression of foreign proteins using thereof (Korean
Patent Publication No. 2000-0034279); Gene coding xylanase of
Bacillus sp. AMX-4 strain and recombinant xylanase through
transformant thereof (Korean Patent Publication No. 2003-0085679);
and Novel Paenibacillus sp. HY-8 strain and xylanase isolated from
it (Korean Patent Publication No. 2007-0082329). However, there was
no specific explanation about the enzyme activity, neither was
reported the cases of using them in domestic industry. Owing to the
advanced technology, new methods to polymerize and produce various
valuable compounds based on biomass such as high molecular
compounds (plastic) have been developed and therefore it is
urgently requested to develop xylanase with novel characteristics
to match with the above.
[0011] For the efficient use of biomass, the development of
saccharification process of cellulose using cellulase and the
development of saccharification process of hemicellulose using
xylanase need to be achieved at the same time. In the enzymatic
saccharification process using xylanase, the characteristics of
each enzyme required for the process are different according to the
pre-treatment method of biomass. For example, in the process of
pre-treatment of biomass using acid, acid-resistant saccharifying
enzyme is required, while alkali-resistant saccharifying enzyme is
required in the process of pre-treatment of biomass using alkali
such as in the processes of pulp or paper production. In the
meantime, heat-resistant enzyme is required for the simultaneous
process of saccharification and fermentation. Most of
commercialized xylanases are suitable for acid condition,
indicating that novel xylanase demonstrating high activity under
alkali condition needs to be developed. It is better to develop
such xylanase that shows high activity in both acid and alkali
conditions. To use xylanase directly in industry, it is important
to secure xylanase gene that is able to maintain high activity
under tough conditions such as high temperature and a wide range of
pH via screening novel microorganisms and enzyme systems, from
which the disadvantages shown in the conventional patents might be
overcome.
[0012] To avoid those xylanases and those strains producing the
same which have already been claimed by other advanced countries,
not to infringe intellectual property right, the present inventors
tried to develop novel xylanase to meet the domestic and further
international need. In the course, the inventors completed this
invention by confirming that the xylanase produced from the novel
Paenibacillus sp. HPL-3 strain demonstrated excellent activity unit
(Unit=mM product/mg protein/min), heat-resistance and a wide range
of optimum pH, compared with the conventional xylanases, confirmed
by solid culture and/or liquid culture enzyme activity measurement
method.
SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to provide xylanase
having high activity at high temperature and in a wide range of
pH.
[0014] It is another object of the present invention to provide a
strain producing the said xylanase.
[0015] It is further an object of the present invention to provide
a polynucleotide encoding the said xylanase.
[0016] It is also an object of the present invention to provide a
recombinant expression vector operably linked to the said
polynucleotide.
[0017] It is also an object of the present invention to provide a
transformant prepared by introducing the said recombinant
expression vector into host cells.
[0018] It is also an object of the present invention to provide a
production method of xylanase containing the step of obtaining
crude enzyme solution by centrifugation after culturing the strain
or the transformant above in a medium.
[0019] It is also an object of the present invention to provide a
xylan decomposer comprising the said xylanase, the said strain, or
the said transformant.
[0020] It is also an object of the present invention to provide a
composition for processing food xylan comprising the said
xylanase.
[0021] It is also an object of the present invention to provide a
feed additive comprising the said xylanase.
[0022] It is also an object of the present invention to provide a
composition for papermaking process comprising the said
xylanase.
[0023] It is also an object of the present invention to provides a
method for decomposing xylan containing the step of adding the said
xylanase, the said strain, or the said transformant to
lignocellulosic biomass or xylan containing solution.
[0024] It is also an object of the present invention to provide a
preparation method of feeds containing the step of adding the said
xylanase, the said strain, or the said transformant to animal feed
materials.
[0025] It is also an object of the present invention to provide the
said xylanase, the said strain, or the said transformant for the
use as a xylan decomposer.
[0026] It is also an object of the present invention to provide the
said xylanase for the use as a composition for processing food
xylan.
[0027] It is also an object of the present invention to provide the
xylanase for the use as a feed additive.
[0028] In addition, it is an object of the present invention to
provide the xylanase for the use as a composition for papermaking
process.
[0029] To achieve the above objects, the present invention provide
the xylanase having the characteristics of (a).about.(d):
[0030] (a) molecular weight: approximately 65 KDa on SDS-PAGE;
[0031] (b) maximum activity in the range of pH 5.about.pH 11;
[0032] (c) maximum activity at the temperature of
50.about.60.degree. C.; and
[0033] (d) xylose production at 50.degree. C., pH 5.about.pH 11: at
least 95 unit/minmg; and xylose production at 60.degree. C., pH
6.about.pH 9: at least 100 unit/mining.
[0034] The present invention also provides a strain producing the
said xylanase.
[0035] The present invention also provides a polynucleotide
encoding the said xylanase.
[0036] The present invention also provides a recombinant expression
vector operably linked to the said polynucleotide.
[0037] The present invention also provides a transformant prepared
by introducing the said recombinant expression vector into host
cells.
[0038] The present invention also provides a production method of
xylanase containing the step of obtaining crude enzyme solution by
centrifugation after culturing the strain or the transformant above
in a medium.
[0039] The present invention also provides a xylan decomposer
comprising the said xylanase, the said strain, or the said
transformant.
[0040] The present invention also provides a composition for
processing food xylan comprising the said xylanase.
[0041] The present invention also provides a feed additive
comprising the said xylanase.
[0042] The present invention also provides a composition for
papermaking process comprising the said xylanase.
[0043] The present invention also provides a method for decomposing
xylan containing the step of adding the said xylanase, the said
strain, or the said transformant to lignocellulosic biomass or
xylan containing solution.
[0044] The present invention also provides a preparation method of
feeds containing the step of adding the said xylanase, the said
strain, or the said transformant to animal feed materials.
[0045] The present invention also provides the said xylanase, the
said strain, or the said transformant for the use as a xylan
decomposer.
[0046] The present invention also provides the said xylanase for
the use as a composition for processing food xylan.
[0047] The present invention also provides the xylanase for the use
as a feed additive.
[0048] In addition, The present invention provides the xylanase for
the use as a composition for papermaking process.
Advantageous Effect
[0049] As explained hereinbefore, the xylanase separated from
Paenibacillus sp. HPL-3, the novel strain of the present invention,
has excellent heat-resistance at 60.degree. C. and demonstrates
excellent xylan decomposing activity in a wide range of pH
(4.about.11), so that this enzyme can be effectively used not only
in the fields of feed, paper and detergent industries, but also in
the saccharification process of lignocellulosic biomass for the
production of raw materials of petroleum alternative material,
performance chemical, and bio-polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0051] FIG. 1 is a electron micrograph illustrating the selected
strain.
[0052] FIG. 2 is a diagram illustrating the selection of active
clones from 1,248 gDNA library.
[0053] FIG. 3 is a diagram illustrating the results of ORF analysis
and homology analysis among ORF amino acids.
[0054] FIG. 4 is a diagram illustrating the activity of the
xylanase separated and purified from the transformant.
[0055] FIG. 5 is a graph illustrating the optimum pH range of the
xylanase.
[0056] FIG. 6 is a graph illustrating the optimum temperature range
of the xylanase.
[0057] FIG. 7 is a diagram illustrating the kinetics of the
separated and purified xylanase.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0058] Hereinafter, the present invention is described in
detail.
[0059] The present invention provide the xylanase having the
characteristics of (a).about.(d):
[0060] (a) molecular weight: approximately 65 KDa on SDS-PAGE;
[0061] (b) maximum activity in the range of pH 5.about.pH 11;
[0062] (c) maximum activity at the temperature of
50.about.60.degree. C.; and
[0063] (d) xylose production at 50.degree. C., pH 5.about.pH 11: at
least 95 unit/min mg; and xylose production at 60.degree. C., pH
6.about.pH 9: at least 100 unit/mining.
[0064] The amino acid sequence of the xylanase of the present
invention preferably contains one of the following sequences, but
not always limited thereto:
[0065] a) the amino acid sequence represented by SEQ. ID. NO:
4;
[0066] b) the amino acid sequence having at least 70% homology,
preferably at least 80%, and more preferably at least 90% homology
with the amino acid sequence represented by SEQ. ID. NO: 4;
[0067] c) the amino acid sequence encoded by the nucleotide
sequence represented by SEQ. ID. NO: 3;
[0068] d) the amino acid sequence of the protein composed of the
modified amino acid sequence having substitution, deletion,
insertion, and/or addition of at least one of amino acids of the
sequence represented by SEQ. ID. NO: 4, which is also identical in
its function to the protein containing the amino acid sequence
represented by SEQ. ID. NO: 4; and,
[0069] e) the amino acid sequence encoded by DNA hybridized with
the DNA containing the nucleotide sequence represented by SEQ. ID.
NO: 3 in the strict condition, which is identical in its function
to the protein containing the amino acid sequence represented by
SEQ. ID. NO: 3.
[0070] The said `strict condition` is determined in the phase of
washing after hybridization. One of the strict conditions is
exemplified as follows: washing with 6.+-. SSC, 0.5% SDS at room
temperature for 15 minutes, then washing with 2.+-. SSC, 0.5% SDS
at 45.degree. C. for 30 minutes, and then washing with 0.2.+-. SSC,
0.5% SDS at 50.degree. C. for 30 minutes twice. More preferable
strict condition herein means washing at a higher temperature. That
is, washing is performed by the same manner as described above only
except that the last washing is performed with 0.2.+-. SSC, 0.5%
SDS at 60.degree. C. for 30 minutes twice. Another example of the
strict condition of the present invention is set with modification
of the above washing process, that is the last two washings are
performed with 0.1.+-. SSC, 0.1% SDS at 65.degree. C. Whoever in
this field can set up and adjust the conditions to satisfy the
strict conditions.
[0071] The xylanase of the present invention demonstrates maximum
activity in the pH range of 5.about.11, preferably in the pH range
of 6.about.10, and more preferably in the pH range of 6.about.9, at
50.about.60.degree. C., but not always limited thereto.
[0072] In a preferred embodiment of the present invention, the
strain having excellent xylan decomposing activity was collected
from the soil containing waste wood residue left after mushroom
cultivation in halfway up the mountain Gara located in Dadae-ri,
Nambu-myeon, Geoje-si, Gyeongsangnam-do, Korea.
[0073] In a preferred embodiment of the present invention, gDNA
library was constructed to separate a gene encoding the enzyme
protein having xylanase activity from the said strain, and then
xylanase activity was examined. From the experiment, one clone was
selected (see FIG. 2), and then nucleotide sequence of the inserted
DNA fragment was analyzed. As a result, the size of the DNA
fragment was 6,956 bp (SEQ. ID. NO: 2) and 11 ORFs were included in
the sequence range of 100 or more amino acids (see FIG. 3). The
present inventors investigated homology of those ORF proteins
encoded by the gene. Then, E. coli was transfected with ORFS (1620
bp, 539 aa) showing 48% homology with endo-1,4-beta-xylanase
(GenBank Accession No: YP.sub.--001817989), and xylanase activity
was investigated. As a result, excellent xylanase activity was
confirmed. Nucleotide sequence of target DNA fragment of the
transformant was also investigated, and as a result ORF of the
novel xylanase gene having the nucleotide sequence represented by
SEQ. ID. NO: 3 was confirmed.
[0074] In a preferred embodiment of the present invention, the
transformant over-expressing xylanase was constructed in order to
produce the novel xylanase having the amino acid sequence
represented by SEQ. ID. NO: 4 in a large scale. Enzyme activity of
the novel xylanase with 65 kD (see FIG. 4) which was produced,
separated, and purified from the said transformant E. coli was
investigated. As a result, maximum activity was observed at
50.about.60.degree. C., in pH range of 4.about.11 (see FIGS. 5 and
6). Even though the activity was inhibited by 74%, 28%, 12%, and
46%, respectively by 1 mM of Cu+2, Zn+2, Fe+2, and EDTA, among
various heavy metal sources, the enzyme activity was hardly
affected at the concentration of 100 HM or rather increased by them
(see Table 1). Enzyme kinetics was also investigated. As a result,
Km, presenting substrate affinity, was 0.2 (see FIG. 7). Hydrolyzed
products were largely xylo-oligomer.
[0075] From the results of sequencing and activity analysis, it was
confirmed that the xylanase produced from the strain identified in
this invention was novel one that was different from the
conventional xylanase.
[0076] The present invention also provides a strain producing the
said xylanase.
[0077] The said strain can be prokaryotic cells including E. coli,
or eukaryotic cells including yeast, animal cells and insect cells,
but not always limited thereto. The said strain producing xylanase
is preferably Paenibacillus sp. HPL-3 deposited under the Accession
No. of KCTC11987BP, but not always limited thereto, and any strain
that is able to produce xylanase can be included in this
invention.
[0078] In a preferred embodiment of the present invention, the
strain having excellent xylan decomposing activity was collected
from the soil containing waste wood residue left after mushroom
cultivation in halfway up the mountain Gara located in Dadae-ri,
Nambu-myeon, Geoje-si, Gyeongsangnam-do, Korea. The collected
strain was identified as a Gram-positive, rod type, non-motile
bacillus without flagellum. The cell size was 1.1 .mu.m and the
cell length was 2.5.about.4 .mu.m (see FIG. 1). The strain had 16S
rRNA represented by SEQ. ID. NO: 1. From the rRNA homology
analysis, it was confirmed that the strain had 95.0% homology with
Paenibacillus sp. CSH12-5 (GenBank Accession No. EF694701) and
95.7% homology with Paenibacillus daejeonensis(T) (GenBank
Accession No. AM141; AF391124). Since no higher homology with any
other strain was confirmed, the strain of the present invention was
named Paenibacillus sp. HPL-3, which was deposited at Korean
Research Institute of Bioscience and Biotechnology in Jul. 20, 2011
under the Accession No. KCTC1198BP.
[0079] The present invention also provides a polynucleotide
encoding the said xylanase.
[0080] The polynucleotide encoding the xylanase of the present
invention preferably contains one of the following sequences, but
not always limited thereto:
[0081] a) the nucleotide sequence represented by SEQ. ID. NO:
3;
[0082] b) the nucleotide sequence having at least 70% homology,
preferably at least 80%, and more preferably at least 90% homology
with the nucleotide sequence represented by SEQ. ID. NO: 3;
[0083] c) the nucleotide sequence encoding the amino acid sequence
represented by SEQ. ID. NO: 4;
[0084] d) the nucleotide sequence encoding the amino acid sequence
of the protein with substitution, deletion, insertion, and/or
addition of at least one of amino acids of the sequence represented
by SEQ. ID. NO: 4, which is also identical in its function to the
protein containing the amino acid sequence represented by SEQ. ID.
NO: 4; and,
[0085] e) the nucleotide sequence comprising DNA sequence to be
hybridized with DNA containing the nucleotide sequence represented
by SEQ. ID. NO: 3 under the strict condition and encoding the
protein having the same function as the protein comprising the
amino acid sequence represented by SEQ. ID. NO: 4.
[0086] The present invention also provides a recombinant expression
vector operably linked to the said polynucleotide.
[0087] In this invention, the nucleotide sequence of the novel gene
encoding the xylanase separated from Paenibacillus sp. HPL-3 strain
was identified, so that a recombinant vector containing the said
gene can be constructed by the conventional method known to those
in the art. The recombinant vector of the present invention can be
a commercialized one, but not always limited thereto, and can be
constructed by the conventional method known to those in the
art.
[0088] The present invention also provides a transformant prepared
by introducing the said recombinant expression vector into host
cells.
[0089] The host cells usable in this invention are not limited, but
preferably selected from the group consisting of prokaryotic cells
including E. coli and bacteria, and eukaryotic cells including
yeast, animal cells and insect cells, and more preferably E. coli,
but not always limited thereto.
[0090] The present invention also provides a production method of
xylanase containing the step of obtaining crude enzyme solution by
centrifugation after culturing the strain or the transformant above
in a medium.
[0091] The production method of the present invention can
additionally include the step of purifying xylanase from the
obtained crude enzyme solution.
[0092] In the above method, the medium is preferably selected among
commercialized media well-known to those in the art that is
considered to be appropriate for the culture of Paenibacillus sp.
HPL-3 (KCTC11987BP) or the transformant of the present
invention.
[0093] To simplify the purification process and to increase
purification efficiency, the present inventors used a specific
resin binding vector for column-chromatography for the construction
of the transformant. It is preferred to select and separate the
enzyme linked to resin in the course of purification. For the
strict condition herein, glutathione binding vector, calmodulin
binding vector, and maltose binding vector can be used and resin
for column filling is determined by considering the vector used. In
this invention, the results obtained by using glutathione binding
vector and resin are presented, but the invention is not limited
thereto.
[0094] The present invention also provides a xylan decomposer
comprising the said xylanase, the said strain, or the said
transformant.
[0095] The xylan decomposer of the present invention can be not
only the xylanase produced in the strain or the transformant of the
invention but also the strain or the transformant itself.
[0096] The present invention also provides a composition for
processing food xylan comprising the said xylanase.
[0097] The present invention also provides a feed additive
comprising the said xylanase.
[0098] The present invention also provides a composition for
papermaking process comprising the said xylanase.
[0099] The composition or the feed additive of the present
invention can include, in addition to the xylanase, one or more
effective ingredients having the same or similar function to the
xylanase. The preferable concentration of the xylanase of the
present invention is 1.about.90% by the total composition or feed
additive, but not always limited thereto.
[0100] Unlike the conventional xylanase, the xylanase of the
present invention demonstrates excellent activity in a wide range
of pH (5.about.11) and temperature (50.about.60.degree. C.) (see
FIGS. 5 and 6). Therefore, the xylanase of the present invention
can be used as the novel xylanase having high activity in the wide
pH range and heat-resistance.
[0101] The use of xylanase in the enzyme market is largely divided
into food industry, feed industry, and technology field (Bedford
and Morgana, World's Poultry Science Journal 52:61-68, 1996). In
food industry (fruit and vegetable production, brewing and liquor
production, bakery and confectionery), xylanase has been used to
improve quality of the product by softening raw materials,
improving purification efficiency, reducing viscosity, and
increasing extraction/filtration efficiency. In feed industry,
xylanase has been used to reduce non-starch carbohydrates in feed
for pig, poultry and ruminants, to improve intestinal viscosity,
and to increase digestibility of protein and starch (Kuhad and
Singh, Crit. Rev. Biotechnol. 13, 151-172, 1993). In addition, in
the field of technology, xylanase has been used in papermaking
process, precisely for biological whitening, reduction of chlorine
decay, saving energy through simplifying mechanical papermaking
process, separation of starch and gluten, production of renewable
fuel (bio-ethanol), and production of chemical raw materials.
[0102] Therefore, it is well understood by those in the art that
the novel xylanase of the present invention can be effectively used
for the decomposition of xylanase industrially used in papermaking
and paper recycling, and in feed and food industry to improve
quality of the products. The composition of the present invention
can be formulated by the conventional method known to those in the
art.
[0103] The present invention also provides a method for decomposing
xylan containing the step of adding the said xylanase, the said
strain, or the said transformant to lignocellulosic biomass or
xylan containing solution.
[0104] In the above method, the amount of the strain, the
transformant, or the novel xylanase produced by the strain or the
transformant can be adjusted by those in the art.
[0105] The present invention also provides a preparation method of
feeds containing the step of adding the said xylanase, the said
strain, or the said transformant to animal feed materials.
[0106] In the above method, the amount of the strain, the
transformant, or the novel xylanase produced by the strain or the
transformant can be adjusted by those in the art.
[0107] The present invention also provides the said xylanase, the
said strain, or the said transformant for the use as a xylan
decomposer.
[0108] The xylan decomposer of the present invention can be not
only the xylanase produced in the strain or the transformant of the
invention but also the strain or the transformant itself.
[0109] The present invention also provides the said xylanase for
the use as a composition for processing food xylan.
[0110] The present invention also provides the xylanase for the use
as a feed additive.
[0111] In addition, The present invention provides the xylanase for
the use as a composition for papermaking process.
[0112] The said xylanase preferably has the characteristics of
(a).about.(d), but not always limited thereto:
[0113] (a) molecular weight: approximately 65 KDa on SDS-PAGE;
[0114] (b) maximum activity in the range of pH 5.about.pH 11;
[0115] (c) maximum activity at the temperature of
50.about.60.degree. C.; and
[0116] (d) xylose production at 50.degree. C., pH 5.about.pH 11: at
least 95 unit/minmg; and xylose production at 60.degree. C., pH
6.about.pH 9: at least 100 unit/min mg.
[0117] The amino acid sequence of the said xylanase preferably
contains one of the below sequences, but not always limited
thereto:
[0118] a) the amino acid sequence represented by SEQ. ID. NO:
4;
[0119] b) the amino acid sequence having at least 70% homology,
preferably at least 80%, and more preferably at least 90% homology
with the amino acid sequence represented by SEQ. ID. NO: 4;
[0120] c) the amino acid sequence encoded by the nucleotide
sequence represented by SEQ. ID. NO: 3;
[0121] d) the amino acid sequence of the protein composed of the
modified amino acid sequence having substitution, deletion,
insertion, and/or addition of at least one of amino acids of the
sequence represented by SEQ. ID. NO: 4, which is also identical in
its function to the protein containing the amino acid sequence
represented by SEQ. ID. NO: 4; and,
[0122] e) the amino acid sequence encoded by DNA hybridized with
the DNA containing the nucleotide sequence represented by SEQ. ID.
NO: 3 in the strict condition, which is identical in its function
to the protein containing the amino acid sequence represented by
SEQ. ID. NO: 3.
[0123] Unlike the conventional xylanase, the xylanase of the
present invention demonstrates excellent activity in a wide range
of pH (5.about.11) and temperature (50.about.60.degree. C.) (see
FIGS. 5 and 6). Therefore, the xylanase of the present invention
can be used as the novel xylanase having high activity in the wide
pH range and heat-resistance for the composition for processing
food xylan, for feed additive, or for papermaking process.
[0124] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples,
Experimental Examples and Manufacturing Examples.
[0125] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1
Isolation and Selection of Strain
<1-1> Isolation of Strain
[0126] The strain of the present invention was collected from the
soil containing waste wood residue left after mushroom cultivation
in halfway up the mountain Gara located in Dadae-ri, Nambu-myeon,
Geoje-si, Gyeongsangnam-do, Korea. Soil sample was collected from
2.about.5 cm under the surface layer, which was dried over wind and
filtered with 2 mm sieve. The filtered soil sample (30 g) was
loaded in 270 ml of sterilized saline (NaCl 8.0 g/l), followed by
shaking in a shaking incubator (37.degree. C., 200 rpm) for 20
minutes. The soil sample stood at room temperature for 30 minutes
to precipitate big soil particles and impurities on the bottom. The
supernatant was transferred in a sterilized container, leading to
the preparation of the first diluted solution. The first diluted
solution was stirred well and then 10 ml of the solution was taken
and loaded in 90 ml of saline to prepare the second diluted
solution (100 ml). The second diluted solution was stirred well and
then 10 ml of the solution was taken again and loaded in 90 ml of
saline to prepare the third diluted solution (100 ml). Then, the
fourth, fifth and sixth diluted solutions were prepared by the same
manner as described above. The 3rd, 4th, 5th, and 6th diluted
solutions were distributed in TSA (Tryptic Soy Agar, Difco Co.)
medium for strain separation, three times by 0.25 ml at a time. The
diluted solutions were smeared evenly on the medium and culture was
performed in a 37.degree. C. bench-type incubator for 2 days. Then,
the formed microorganism colonies were selected. The selected
colonies were separated by shape, size, color, and other relevant
factors. The separated colonies were sub-cultured in TSA medium, to
isolate pure strains which were stored at -70.degree. C. for being
used as the mother strain.
<1-2> Selection of Strain
[0127] For the selection of active strain showing xylanase activity
among the isolated pure strains, soft agar double medium was
prepared by adding birch xylan (Fluka Bio Chemika. Co.) to TSA
medium by 0.5.about.1.0%, to which the isolated strains were
inoculated. After overnight culture, those strains forming
translucent zone (halo) around the cultured colony and active
clones were selected by Congo-red staining method (Theater R M, PJ.
Wood. Appl Environ Microbiol 43, 777-780, 1982; Beguin P.
Analytical Biochemistry, 131(2):333-336, 1983). Xylan decomposing
activity of the selected strain was measured again to confirm
reproducibility. Among those strains, the strain demonstrating most
excellent xylan decomposing activity was selected finally as the
microorganism producing xylanase.
Example 2
Identification of Strain
[0128] The present inventors cultured the strain producing xylanase
demonstrating the highest activity, isolated in Example 1, at
30.degree. C., and then performed Gram staining and spore staining.
As a result, the strain of the present invention was confirmed to
be Gram-positive bacillus forming spores. The morphology of the
strains was observed under electron microscope. As shown in FIG. 1,
the strain was identified as a rod type, non-motile bacillus
without flagellum. The cell size was 1.1 .mu.m and the cell length
was 2.5.about.4 .mu.m. From analysis of 16S rRNA nucleotide
sequence of the strain, 1234 bp rDNA represented by SEQ. ID. NO: 1
was obtained, followed by screening with GenBank database. As a
result, it was confirmed that the strain had 96.4% homology with
Pantoea agglomerans ZFJ-15 (GenBank Accession No. EU931554) and
96.3% homology with Paenibacillus sp. WPCB158 (GenBank Accession
No. FJ006910). Since no higher homology was confirmed, the strain
of the present invention was named Paenibacillus sp. HPL-3, which
was deposited at Korean Research Institute of Bioscience and
Biotechnology in Jul. 20, 2011 under the Accession No. of
KCTC1198BP.
Example 3
Separation of Novel Xylanase
<3-1> Construction of Paenibacillus Strain Gene Library and
Activity Test
[0129] To separate the gene encoding the enzyme protein having
xylanase activity from the Paenibacillus sp. HPL-3 strain separated
and identified in Example 1 and Example 2, genome DNA was first
extracted to construct gDNA library containing gene fragments less
than 5 kb. To construct the library, the extracted DNA was
fragmented into 1.about.6 kb sized fragments by random digestion,
followed by electrophoresis on agarose gel. The fragments were
sorted by size and those DNA fragments having approximately 5 kb
size were selected. The fragments were inserted in pCB31 plasmid
vector, with which E. coli DH10B was transfected. Xylanase activity
was investigated with 1,248 clones of the constructed library in
solid or liquid condition.
<3-2> Xylanase Activity Test
[0130] The measurement of xylanase activity (xylan decomposing
activity) of the isolated strain, active clone, transformant, and
isolated/purified enzyme was performed by either or both of the
following two methods. The first method was enzyme activity
measuring method using solid culture. Particularly, soft agar
double medium was prepared by adding birch xylan (Fluka Bio
Chemika. Co.) to LB medium by 0.5.about.1.0%, to which the strain
was inoculated, followed by culture for overnight. On the next day,
the strain and active clone forming translucent zone (halo) around
the cultured colony were selected by Congo-red staining. The second
method was enzyme activity measuring method using liquid culture,
that is DNS (3,5-dinitrosalicylic acid) quantitative method (Miller
G. L. Anal Chem 31, 426-428, 1959). Particularly, 50 .mu.l of
substrate solution (50 mM Tris-HCl containing birch xylan by 2%, pH
7.0) was added to 50 .mu.l of enzyme solution, followed by reaction
at 50.degree. C. for 20 minutes. 200 .mu.l of DNS solution was
added thereto, followed by further reaction at 90.degree. C. for 5
minutes. Then, OD.sub.540 was measured. 1 unit of the enzyme was
defined as the enzyme activity of 1 mg xylanase to produce 1 pmol
of reducing sugar (xylose) per 1 minute. One clone showing the best
activity, GM3-SLX1, was selected by liquid culture and solid
culture activity test (FIG. 2).
<3-3> Selection of Xylanase Active Clone and Gene Assay
[0131] Sequencing of the DNA fragment inserted in the clone
selected in Example <3-2> was performed. As a result, the
size of the DNA fragment inserted in the plasmid was 6,956 bp (SEQ.
ID. NO: 2). ORF (Open Reading Frame) was also investigated by using
NCBI Blast P or Blast N program (//www.ncbi.nlm.nih.gov/). Among
the ORFS analyzed above, 11 ORFS having the size of at least 100
amino acids were named as follows: SLX-O1, SLX-O2, SLX-O3, SLX-O4,
SLX-O5, SLX-O6, SLX-07, SLX-O8, SLX-O9, SLX-O10, and SLX-O11. Among
the ORFs, SLX-O9 (SEQ. ID. NO: 3) showing 48% homology with
endo-1,4-beta-xylanase (AJ006646) was selected as a target. Based
on nucleotide sequence of the target, the primer having the
addition of XhoI and BamHI sites was constructed, followed by
amplification by PCR. Then, the amplified primer was inserted in
pGEM-T-Easy vector (Promega) to construct a recombinant
plasmid.
[0132] Particularly, 1 ng of GM3-SLX1 template plasmid was mixed
with the primer set (10 pmol) composed of the forward primer
represented by SEQ. ID. NO: 5
(5'-CTCGAGATGGATACATTGAAGTTGTATGTG-3') and the reverse primer
represented by SEQ. ID. NO: 6 (5'-GGATCCCTATTCGTTGCTCCCC-3'),
followed by PCR using PCR Premix (GenetBio) as follows:
predenaturation at 94.degree. C. for 5 minutes, denaturation at
94.degree. C. for 30 seconds, annealing at 55.degree. C. for 30
seconds, extension at 72.degree. C. for 1 minute, 30 cycles from
denaturation to extension, and final extension at 72.degree. C. for
7 minutes. Then reaction was terminated at 4.degree. C. The PCR
amplified product was purified by using GENCLEAN II Kit
(Q-Biogene), and a recombinant DNA was constructed with pGEM-T-easy
vector using T4 ligase (RBC). E. coli transformant was prepared by
transfecting E. coli JM109 with the recombinant plasmid. The
transformant was cultured in LB liquid medium, and then, plasmid
DNA was extracted by using HiYield.TM. Plasmid Mini Kit (RBC). The
extracted plasmid DNA was digested with XhoI (NEB) and BamHI (NEB),
followed by electrophoresis to confirm the insertion of the target
DNA fragment. Xylanase activity of the said transformant was
investigated in the liquid phase as explained in Example
<3-2>. As a result, xylanase activity was observed
therein.
[0133] Nucleotide sequence of the target DNA fragment of the
transformant was investigated and as a result ORF of the novel
xylanase gene represented by SEQ. ID. NO: 4 was confirmed.
<3-4> Construction of Transformant Over-Expressing Xylanase
(pIVEX-GST-PX3)
[0134] To construct a transformant over-expressing the novel
xylanase, PCR was performed using GM3-SLX1 plasmid as a template
with the primer set composed of the sequence represented by SEQ.
ID. NO: 5 and the sequence represented by SEQ. ID. NO: 6. PCR
reaction mixture and the reaction condition were same as described
in Example <3-2>. The amplified product was purified and
digested with XhoI (NEB) and BamHI (NEB), which was inserted in
pIVEX-GST (Roche), the protein over-expressing vector, to construct
the recombinant over-expression plasmid. E. coli transformant
over-expressing xylanase was prepared by transfecting E. coli BL21v
(RBC) with the recombinant plasmid. The constructed E. coli
transformant over-expressing xylanase was cultured and plasmid DNA
was extracted. The extracted plasmid DNA was digested with the said
restriction enzymes, followed by electrophoresis to confirm that
the target DNA and the vector were successfully recombinated. The
identified strain was cultured in LB liquid medium (supplemented
with 100 ampicillin/ml) for 18 hours (37.degree. C., 250 rpm,
A600=1.0) and inoculated in fresh LB liquid medium. When OD.sub.600
reached 0.4.about.0.6, the medium was treated with 1 mM of IPTG,
followed by further culture for 3 hours. Then, cells were
collected, resuspended and sonicated. The sonicated cell suspension
was centrifuged at 10,000 g to separate supernatant and
precipitate. The target protein over-expressed in the supernatant
was confirmed. Molecular weight of the target protein was
approximately 65 kD, which was confirmed by SDS-PAGE (FIG. 4).
<3-5> Condition for Xylanase Activity Expression
[0135] In this example, pH, temperature, and metal ion-dependent
activity of xylanase over-expressed and isolated from the E. coli
transformant over-expressing the novel xylanase constructed in
Example <3-4> was investigated by the same manner as
described in example 3-2. pH of the reaction solution was regulated
to pH 4.about.5 with citric acid buffer, to pH 6.about.8 with
phosphate buffer, to pH 7.about.9 with Tris/HCl buffer, and to pH
9.about.11 with glycine/NaOH buffer. To investigate the effect of
metal ions on xylanase activity, CaCl.sub.2, MgCl2, MgCl.sub.2,
CuCl.sub.2, ZnCl.sub.2, and FeCl.sub.3 were added by 1 mM each. It
was further investigated how other salts such as NaCl, LiCl, KCl,
NH.sub.4Cl, EDTA, CsCl.sub.2, 2-ME (2-Mercaptoethanol), DTT
(Dithiothreitol), PMSF (Penylmethylsulfonyl fluoride), acetate, and
furfural could affect xylanase activity.
[0136] As a result, the novel xylanase demonstrated the maximum
activity at the pH range of 4.about.11 as shown in FIG. 5, and at
the temperature range of 50.about.60.degree. C., as shown in FIG.
6. As shown in Table 1, xylanase activity was inhibited by 74%,
28%, 12%, and 46% respectively by 1 mM of such heavy metal or
additive as Cu.sup.+2, Zn.sup.+2, Fe.sup.+2, and EDTA.
TABLE-US-00001 TABLE 1 Relative activity according to conc. of
additive (%) Additive 1 mM Negative Control 100 NaCl 105 LiCl 101
KCl 101 NH4Cl 99 CaCl.sub.2 102 MgCl.sub.2 93 MnCl.sub.2 98
CsCl.sub.2 97 CuSO.sub.4 26 ZnSO.sub.4 72 FeCl.sub.3 88 EDTA 54
2-ME 89 DTT 95 PMSF 95 Acetate 101 Furfural 98
Example 4
Mass-Production of the Novel Xylanase
[0137] E. coli BL21-Gold (DE) (Stratagene, USA) was transfected
with pIVEX GST-xylanase recombinant vector (Bioprogen Co., Ltd.,
Korea) containing the gene (SEQ. ID. NO: 3) encoding the novel
xylanase represented by SEQ. ID. NO: 4, which was inoculated in
liquid medium (LB 25 g/L) supplemented with ampicillin (50
.mu.g/ml), followed by shaking culture at 37.degree. C. at 150 rpm
until OD.sub.603 reached 0.4.about.0.6. To induce the expression of
the target protein in E. coli cells, IPTG
(isopropyl-D-thiogalactoside) was added to the suspension at the
concentration of 1 mM, followed by further culture for hours. The
culture solution was centrifuged at 10,000 rpm for 10 minutes and
the recovered precipitate was washed with PBS twice. The washed
precipitate was re-suspended in PBS and then cell were lysed by
using a ultrasonicator (Cosmo Bio Co., LTD). Centrifugation was
performed (12,000 rpm, 10 minutes) to obtain supernatant. To
isolate xylanase from the supernatant, glutathione S-transferase
column (GST binding resin column, Novagen) was used. For xylanase
separation, the obtained supernatant was filled in glutathione
S-transferase column (GST binding resin column, Novagen)
equilibrated with washing buffer (50 mM Tris-HCl, 100 mM NaCl; pH
7.0), followed by treatment of factor Xa protease (NEB) and
separation using washing buffer (50 mM Tris-HCl, 100 mM NaCl; pH
7.0). The enzyme activity of xylanase in each sample recovered from
the purification stage was investigated. The purification of active
fraction showing the enzyme activity was confirmed by SDS-PAGE.
Protein content was measured by using Bradford method (Bradford,
Sigma Aldrich), and BSA (bovine serum albumin) was used as the
standard protein.
[0138] As a result, it was confirmed that xylanase was
mass-produced easily by glutathione resin column chromatography.
Considering the condition of xylanase conjugated onto the resin, it
was confirmed that xylanase activity was not changed. Therefore,
the novel xylanase could be used for high efficiency conversion
process via enzyme immobilization method. The enzyme purified by
the above method demonstrated at least 5 times higher activity than
before purification. The enzyme activity at 50.degree. C. was
97.37, 124.19, 122.21, 124.61, 122.95, 103.27, and 96.08 units,
respectively at pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0.
and pH 11.0, suggesting that the enzyme activity was guaranteed in
a wide range of pH. In the meantime, the enzyme activity at
60.degree. C. was 26.93, 105.19, 123.56, 120.95, 112.25, 29.89, and
22.76 units (.mu.M of xylose produced by 1 mg of enzyme per 1
minute), respectively at pH 5.0, pH 6.0, pH 7.0, pH 8.0. pH 9.0, pH
10.0, and pH 11.0, suggesting that the enzyme had excellent
heat-resistance and excellent activity.
Example 5
Characteristics of the Mass-Produced Noble Xylanase
[0139] To investigate the characteristics of the enzyme, the
purified xylanase was loaded in a test tube, to which 50 mM
Tris-HCl (pH 7.0) buffer containing birch xylan at different
concentrations was added. The mixture was reacted at 50.degree. C.
for 20 minutes to investigate enzymatic reaction rate
(Lineweaver-Burk).
[0140] As a result, as shown in FIG. 7, affinity K.sub.m value to
xylan substrate was 0.2. Most of the hydrolyzates were xylose and
xylo-oligomer. Xylanase activity against other xylans (beech, oats,
etc) was similar to that against birch xylan.
INDUSTRIAL APPLICABILITY
[0141] As explained hereinbefore, unlike the conventional xylanase,
the novel xylanase of the present invention shows high activity in
a wide range of pH and excellent heat-resistance to decompose
xylan, the major component of various lignoceilulosic biomass, so
that it can not only be used for the preparation and development of
a xylan decomposer, a composition for food processing, a feed
additive, or a composition for papermaking process, but also
contribute to bio-chemical industry via the application to the
development of bio-fuel, alternative material, performance
chemical, and bio-polymer.
[0142] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
Claims.
Sequence CWU 1
1
611393DNAPaenibacillus sp. 1ggctcaggac gaacgctggc ggcgtgccta
atacatgcaa gtcgagcggg gttatgttaa 60aagcttgctt ttaacataac ctagcggcgg
acgggtgagt aacacgtagg caacctgccc 120atcagactgg gataactacc
ggaaacggta gctaataccg gatacatcct ttccctgcat 180ggggagagga
ggaaagacgg agcaatctgt cactgatgga tgggcctgcg gcgcattagc
240tagttggtgg ggtgaaggcc taccaaggcg acgatgcgta gccgacctga
gagggtgatc 300ggccacactg ggactgagac acggcccaga ctcctacggg
aggcagcagt agggaatctt 360ccgcaatggg cgaaagcctg acggagcaac
gccgcgtgag tgatgaaggt tttcggatcg 420taaagctctg ttgccaggga
agaacgtctt gtagagtaac tgctacaaga gtgacggtac 480ctgagaagaa
agccccggct aactacgtgc cagcagccgc ggtaatacgt agggggcaag
540cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ctctttaagt
ctggtgttta 600atcccgaggc tcaacttcgg gtcgcactgg aaactgggga
gcttgagtgc agaagaggag 660agtggaattc cacgtgtagc ggtgaaatgc
gtagatatgt ggaggaacac cagtggcgaa 720ggcgactctc tgggctgtaa
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt 780agataccctg
gtagtccacg ccgtaaacga tgaatgctag gtgttagggg tttcgatacc
840cttggtgccg aagttaacac attaagcatt ccgcctgggg agtacggtcg
caagactgaa 900actcaaagga attgacgggg acccgcacaa gcagtggagt
atgtggttta attcgaagca 960acgcgaagaa ccttaccagg tcttgacatc
cctctgatcg gtctagagat agatctttcc 1020ttcgggacag aggagacagg
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080gttaagtccc
gcaacgagcg caacccttat gcttagttgc cagcaggtca agctgggcac
1140tctaagcaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa
atcatcatgc 1200cccttatgac ctgggctaca cacgtactac aatggccggt
acaacgggaa gcgaaagagc 1260gatctggagc gaatcctaga aaagccggtc
tcagttcgga ttgcaggctg caactcgcct 1320gcatgaagtc ggaattgcta
gtaatcgcgg atcagcatgc ccgcggtgaa tacgttcccg 1380ggtcttgtac aca
139326956DNAPaenibacillus sp. 2catcctctct ataagcttaa cagaagccta
gcccaataga caaaagacgg gtataccgtc 60cccggtcccg tcttttctta tcatgaatcg
ctgctgtata tgccaggcga gctggaagtg 120aattgcgctt caaccttatt
tcttggttgt tgttactact ttagctgctg cttttggagc 180tgttttcact
gctgtttttg gagctgtttt taccactgtt tttgctggag tcactttttt
240aacaggagct ttgtgagccg cttttttagc tacaacatgg tgtgcacgag
ttttgtgagc 300tgttgcttta tgttttactg cataatgtct tttagcatga
tgtttaacgg tacggtgctt 360cgcttttaca gcttttgctt tatgtgccgt
ttttttggca acagcttttt tggcaggagc 420ttttttagct gttgttactt
tatgagctgt tgcagctttt ttaacaggtg ctttcaccac 480tggtgttttc
gtcgcaggtg cttttactgc tggtgctttg gtagccgggg tcgtaaccgc
540cggtgctttc gttgctggtg cagttgtagc tggcgttgtt gtcgctgtag
cggcgaatgc 600actgcttgct cctcccaata cgcttactac ggtcaatacg
gctgctgcat ttttagtcca 660ttttttcatg attgtatcac ctctccttta
aagtatcttt agcttatcag gagaattcgg 720gataattgtg gaacaaatgt
gtgaaaagta tatgaaaaag cgtgcagccc cgtattcaaa 780aaatacgtga
gtctgcacgc tgtacgtttt acacaattac attcactaac cgatggcaat
840ccaggaaaca ataccgtagt tgtgcgcggt ttcgttccat ttggttacta
caatcaccgc 900tccattcggc gtctgcgatt tcagcgtcgc atgaaagaga
ggatggttcg tcatcgctac 960cagcgtatag tccgggttca taaaaggctc
tgcgaatata atgatcactt ccgtctctcc 1020ttcgctgccc ttcaaaagaa
acggcgctct gccaaactgc tgcaatgcgg actgattggc 1080acaagtacga
accggggtga tgctcagatg ctccggcttc actgcgttca gctccagctc
1140acgcgagcct acggagcctt ccaccagatg atgcccttca atgctttgct
ctgccaagtg 1200atggctttgt accgcagcgg tctgtagatg ctccgatcct
acagcttccg ggctcagcac 1260ctcgctgtcg acggcctgag ctttcagatg
gctgcgagat acactatctg cctggagtct 1320gtcgccactg ataccttcct
ccggcaaaag ccccgagaga tgaatgtccg tggacaactg 1380agccagcgtc
actgcgcccg gcttcaggtg gtgcgcatca atggcttctg ctccaatgtg
1440tcggccttca atggcttcct cagccacatg ctgggactgt accgctccat
ttgcaagctt 1500gccggacgta accgcttctt cttgcaatgt gtcttccgac
accacctgcc agcctatatg 1560ctgcggctgg atggcacctg cttgcagatg
ctccgactgg acggaatcca aggcgatatg 1620ccttgattgc accgcgcctt
ctgccagcgc cgattcctgt accgccccat cggccagatg 1680aaggtggtgt
accgcttgct ccaccagatg tacggagctt acgctggctt ccgccaaatt
1740cgaagcacgc accgcccgaa gcgcaagctt ctcagcattc acgctcccgg
cctggagcgc 1800gtgaacagat acacttcccg gcgcaatatg ccgggtcatc
actgctgaag cctgcaaatg 1860gtcagaggtg acggcttccg gggccaaatg
gctcgattgg atcgcctctt ctgccaaatg 1920gttcccttgc accgcttcgt
ccgtaatgtg ccatggctgt accgagtggg cggacagctt 1980ggccggccgt
cacgctctca tccgccagtt tgccggacgt aacagactca tcctgcaagg
2040catcggtcga tatcgcctgt gatgcgatat gctcgctctg aatggttccg
atgtgcagat 2100gatgtggctg gatggactcc ggtgcgatat gcacggactt
cactgccccc tctgccagcg 2160ctctgtcctg taccgctcca tctgccagat
gccgggggtg tacggcctgc gcagacaaat 2220gtgaagggcc gacgatgcct
tccgcgaggt tggtcgcttg cacggcccga gccgccagct 2280tctccgaggt
gacgctttcc gcttgaagcg cacgaccgga tacgctatcc ggggccagat
2340gtctcgtaag caccgccgaa gcctgcaaat ggtcagaggt gacggcttcc
ggggccaaat 2400ggctcgattg gatcgcttct tctgccaaat ggttcccttg
caccgcttcg tccgtaatgt 2460gccatggctg taccgagtga gcggacaact
tggcggccgt cacactctca tccgccagtt 2520tgtcagacgt aacagactca
tcctgcaagg catcggtcga tatcgcctgt gatgcgatat 2580gctcgccctg
aatggttccg atgtgcagat gatgtggctg gatggactcc ggtgcgatat
2640gcacggactt tactgcccct tctgccagcg ctctgtcttg taccgctcca
gctgccaaat 2700gccgggggtg tacggcctgc gcagacaaat gtgaagggcc
gacgctgcct tccgcgagat 2760tcgtcgcttg caccgagcgt gctgccagct
tttccgaggt gatgctttcc gcttgaagcg 2820cacgatcgga tacactatcc
ggggccagat gtctcgtatg caccgacgaa gactgtaaat 2880gaaccgaggt
gaccgcttcg gctgccagat gtgtcgaatc cacagccgcc acagccaggt
2940gactgctttg tacagcttca tctgtaatat gctcggactg cactgcatgg
gcagatagct 3000tgatggaggt tacacttcct ccgccagttt gtcggacgta
acagactcat tctgcaaagc 3060atcggtcgat atcgcctgtg atgtgatatg
ctcgccctga atggttccga tgtgcagatg 3120gtgcggctgg atagactccg
gtgcgatgtg cacggacttc actgccccct ctgccagcgc 3180tctttcctgt
accgctccaa ctgctagatg ccgggggtgt acggcctgcg cagataaatg
3240cggaggaccc acgctgcctt ccgcgaggtt cattccttgt accgagcgcg
ccgccagctt 3300ttccgaggta atgctttctg cttgaagcgc acgaccggat
acgctatccg gggccagatg 3360tctcgtaagc accgtggagg cttgtagatg
taccgaggtg accgctccgg ccgccagatg 3420cgccgaatcg acagccgcca
cagccagatg gttgcttctc ctgattcgtg agctggggat 3480gcttggaagc
ttctgggtgt atatcgtgcc tgggctgatc ggggtgttca atgtgattgt
3540aattcgctcc tttattgagg gcttaccgga agggatcttg gagtcggcgc
gtatagatgg 3600ggcgggggag tttgcgactt ttatgcgtat cgttttgccg
ctatgtgtcc cggtgctggc 3660aacggtgtcg ctgttcacgg cagtagcgca
atggaactcc tggttcgatg tatttttgta 3720caattcgtcg tatgagcagt
ggagtaccct ccagtatgag ctgatgaaaa tattgcaaaa 3780ttccaacacg
tccgtgaacg ctcaggatta tgccagccaa ttcgcaggct cggaaaatct
3840ggcgaaggcg gttactccta cctccattcg tgccacgatg acaattgtgg
cgtctgtgcc 3900tattattttg gtctatccat ttttgcagaa atattttgtg
aagggtatga ctttgggcgg 3960tgtcaaggga taagcaagga ccggaaatgt
gtgaaactcg ctttcttata ttttgaaaat 4020atgggcgtag gagggattgc
gttgaggtca tttttgtata aatctttggg tctggtgctg 4080gtcggggtgt
tgttattgcc agtggggtgg tgggggctgt ctgtagcgga ggcgactcca
4140actgtggaac attcccagtc agttgcggat acggttctgt cgacggggag
cgtgaataag 4200accatcggga cgtatggatt tgagcaaggc aacgttgagg
gctggaagcc tcgggggact 4260tatacccaaa tcgcaaccgt ctcagaagct
gcatatggcg gcgtacacag cctgaaggta 4320accgctcgca cggaggtttg
gaacggtgcg gagctggatg tgaagtcgct gttgcagccg 4380ggtgtggaat
atgagattag cggctatgtg aagcaggacg gtaattctac gacgccgagc
4440gtgattaagt ttacggtgga gcagcagccg acaggcgggg ccacgacatg
gaagacggtt 4500gcgcagacag agacgacgga tacatcgtgg gccaaacttc
aagggacata tacattcacc 4560ggggggatgg atacattgaa gttgtatgtg
gaaagctcga atcctgcaca ggcctattat 4620ctggatgagg tggaaatcag
gcaggtgtcg gagacaccaa ccactccccc aacggagccg 4680acaagtggta
tcgaatccag atttgaagat ggtacggctc aaggctgggt atcccgtatg
4740ggcaccgaga cggtgcaggt gtcgaatgcc gatgcacgaa ccggatcgta
cagtctgttg 4800acgacaggca gacaacaaac atatgccggg ccaaagctgg
acgtgactgc tacagtgcaa 4860aaaggcagcc gttacacggt cagcgcttgg
gtaaagctgg caccgggcga gcagcagcct 4920gccaaggtgc gcctcagcgt
acagcgcgat catcaaggtg aaagtacgta tgaaactgtg 4980gtaggcaaca
cggccatcac gactggggga tggacgcatt tgtacggaac gtatactctc
5040gcgcatgagg cagacaccgt ctctatgtat ttggaaacgc cagaaggtac
ggcttccttt 5100tacatggatg atttcgagct gtcgcttgtg ccgccgttgg
ctattgagaa ggacattccc 5160tctttgcatg gattgtatca gggacagttc
agcatcggta cggctattga agctttccag 5220acagaagggg cttacgggga
gctggtgcag aagcatttta acagcgtcgt cgccggaaat 5280gcgatgaagc
cgatctcctt gcagccgtcc gaaggacagt tccattggga agaagcagac
5340cggattgtgc aatttgccca gcagcatggg attgctatcc gtttccatac
actggtgtgg 5400cataaccaga ccggcgattg gatgtttaag gataagaatg
gacagccgat gacgccgacc 5460gcggaaaata aaaagctttt gctggatcgg
ctggagacgc atattcgtgc tgttgcagcc 5520cgttataaaa atgtaatcac
cgattgggat gtggtgaatg aagtcattga tcccgaccag 5580ccggacggta
tgcgccgcag caagtggtat cagattaccg gcacggatta tattgacaaa
5640gcattccgtg tcacgaggga agcggcgggt ccgaacgccc ggctgtacat
taacgattac 5700aacacgcatg aaccgaagaa acgggatttt ctgtacaatt
tggtgcgtga tttactcgct 5760aaaggcgtac cgatcgatgg cgtaggtcac
caatcgcata tccgcctgga gttccctgct 5820attgacgaga tggagcagtc
tattgaaaag tttgcttcgc tcggcctgga taatcagatc 5880acggagctgg
atatgggcct gtattctaat gatacagacc actacgaaac gatacctgaa
5940gccatgctga tccggcaggc tcaccgctat cgggcgttgt tcgatatgtt
ttcccgacag 6000caggagcata tcagtaatgt gacgatttgg ggtacggatg
atggaaatac gtggctcagt 6060atgttcccga ttgcccggct ggataagccg
ctgctgttcg acgaacggtt aaaggccaaa 6120tatgcctatt gggcgcttgt
tgacccgtcc aaagtaccgc cgctgccagc ggggagcaac 6180gaatagcaac
ataggtatgg attacacgtg gaaatcattt ctatacacca aaaaggcagg
6240ccgcctctga catgccacaa ggacatgcga aggacgatct gccttttact
gtaaaataat 6300acgatctcta ctccagattg gcgtgttgac caaacataga
ttcagaagtc agcttgcgta 6360gctccatcgt tctgagcacc aatgcatcgc
catacagcag caaggtttgc tcaaatagtg 6420aacccatggg ctgaatggtt
tggtaatcct tgttggatgg atccttgggt gcgcccggaa 6480gcctgacgat
aatatcagcc agcttgccaa tggtcgatcc aggggaggtg gtcagaagtg
6540ccagagaagc ccccagcttt ttcgtctttt ctgccatcga ggtcaaactt
ttggtttcac 6600cagagcctga accgataatc agcaagtcac cttcgcccaa
acctggagtt actgtttccc 6660cgaccacata agcctgaaca cccatatgca
tcagccgcat cgccagcgaa cggatcatga 6720atccagaacg gcctgcgcct
gcgacgaaca ccttattcgc tgaggtaatg gactggatca 6780gttgctctga
ttcctcatca ttgattagct gtggaaccaa ttgcagttcc ttgagcacct
6840cggataaata ttgagaggtt tccattggaa ttaaccttgt ttaatgagct
gctgcatttc 6900ggaagcaacg gcctttttgt cgtcctcacc agtgatcccg
ccgccaacaa taacca 695631620DNAArtificial SequenceSLX-O9 3atggatacat
tgaagttgta tgtggaaagc tcgaatcctg cacaggccta ttatctggat 60gaggtggaaa
tcaggcaggt gtcggagaca ccaaccactc ccccaacgga gccgacaagt
120ggtatcgaat ccagatttga agatggtacg gctcaaggct gggtatcccg
tatgggcacc 180gagacggtgc aggtgtcgaa tgccgatgca cgaaccggat
cgtacagtct gttgacgaca 240ggcagacaac aaacatatgc cgggccaaag
ctggacgtga ctgctacagt gcaaaaaggc 300agccgttaca cggtcagcgc
ttgggtaaag ctggcaccgg gcgagcagca gcctgccaag 360gtgcgcctca
gcgtacagcg cgatcatcaa ggtgaaagta cgtatgaaac tgtggtaggc
420aacacggcca tcacgactgg gggatggacg catttgtacg gaacgtatac
tctcgcgcat 480gaggcagaca ccgtctctat gtatttggaa acgccagaag
gtacggcttc cttttacatg 540gatgatttcg agctgtcgct tgtgccgccg
ttggctattg agaaggacat tccctctttg 600catggattgt atcagggaca
gttcagcatc ggtacggcta ttgaagcttt ccagacagaa 660ggggcttacg
gggagctggt gcagaagcat tttaacagcg tcgtcgccgg aaatgcgatg
720aagccgatct ccttgcagcc gtccgaagga cagttccatt gggaagaagc
agaccggatt 780gtgcaatttg cccagcagca tgggattgct atccgtttcc
atacactggt gtggcataac 840cagaccggcg attggatgtt taaggataag
aatggacagc cgatgacgcc gaccgcggaa 900aataaaaagc ttttgctgga
tcggctggag acgcatattc gtgctgttgc agcccgttat 960aaaaatgtaa
tcaccgattg ggatgtggtg aatgaagtca ttgatcccga ccagccggac
1020ggtatgcgcc gcagcaagtg gtatcagatt accggcacgg attatattga
caaagcattc 1080cgtgtcacga gggaagcggc gggtccgaac gcccggctgt
acattaacga ttacaacacg 1140catgaaccga agaaacggga ttttctgtac
aatttggtgc gtgatttact cgctaaaggc 1200gtaccgatcg atggcgtagg
tcaccaatcg catatccgcc tggagttccc tgctattgac 1260gagatggagc
agtctattga aaagtttgct tcgctcggcc tggataatca gatcacggag
1320ctggatatgg gcctgtattc taatgataca gaccactacg aaacgatacc
tgaagccatg 1380ctgatccggc aggctcaccg ctatcgggcg ttgttcgata
tgttttcccg acagcaggag 1440catatcagta atgtgacgat ttggggtacg
gatgatggaa atacgtggct cagtatgttc 1500ccgattgccc ggctggataa
gccgctgctg ttcgacgaac ggttaaaggc caaatatgcc 1560tattgggcgc
ttgttgaccc gtccaaagta ccgccgctgc cagcggggag caacgaatag
16204539PRTArtificial SequenceSLX-O9 protein sequence 4Met Asp Thr
Leu Lys Leu Tyr Val Glu Ser Ser Asn Pro Ala Gln Ala 1 5 10 15 Tyr
Tyr Leu Asp Glu Val Glu Ile Arg Gln Val Ser Glu Thr Pro Thr 20 25
30 Thr Pro Pro Thr Glu Pro Thr Ser Gly Ile Glu Ser Arg Phe Glu Asp
35 40 45 Gly Thr Ala Gln Gly Trp Val Ser Arg Met Gly Thr Glu Thr
Val Gln 50 55 60 Val Ser Asn Ala Asp Ala Arg Thr Gly Ser Tyr Ser
Leu Leu Thr Thr 65 70 75 80 Gly Arg Gln Gln Thr Tyr Ala Gly Pro Lys
Leu Asp Val Thr Ala Thr 85 90 95 Val Gln Lys Gly Ser Arg Tyr Thr
Val Ser Ala Trp Val Lys Leu Ala 100 105 110 Pro Gly Glu Gln Gln Pro
Ala Lys Val Arg Leu Ser Val Gln Arg Asp 115 120 125 His Gln Gly Glu
Ser Thr Tyr Glu Thr Val Val Gly Asn Thr Ala Ile 130 135 140 Thr Thr
Gly Gly Trp Thr His Leu Tyr Gly Thr Tyr Thr Leu Ala His 145 150 155
160 Glu Ala Asp Thr Val Ser Met Tyr Leu Glu Thr Pro Glu Gly Thr Ala
165 170 175 Ser Phe Tyr Met Asp Asp Phe Glu Leu Ser Leu Val Pro Pro
Leu Ala 180 185 190 Ile Glu Lys Asp Ile Pro Ser Leu His Gly Leu Tyr
Gln Gly Gln Phe 195 200 205 Ser Ile Gly Thr Ala Ile Glu Ala Phe Gln
Thr Glu Gly Ala Tyr Gly 210 215 220 Glu Leu Val Gln Lys His Phe Asn
Ser Val Val Ala Gly Asn Ala Met 225 230 235 240 Lys Pro Ile Ser Leu
Gln Pro Ser Glu Gly Gln Phe His Trp Glu Glu 245 250 255 Ala Asp Arg
Ile Val Gln Phe Ala Gln Gln His Gly Ile Ala Ile Arg 260 265 270 Phe
His Thr Leu Val Trp His Asn Gln Thr Gly Asp Trp Met Phe Lys 275 280
285 Asp Lys Asn Gly Gln Pro Met Thr Pro Thr Ala Glu Asn Lys Lys Leu
290 295 300 Leu Leu Asp Arg Leu Glu Thr His Ile Arg Ala Val Ala Ala
Arg Tyr 305 310 315 320 Lys Asn Val Ile Thr Asp Trp Asp Val Val Asn
Glu Val Ile Asp Pro 325 330 335 Asp Gln Pro Asp Gly Met Arg Arg Ser
Lys Trp Tyr Gln Ile Thr Gly 340 345 350 Thr Asp Tyr Ile Asp Lys Ala
Phe Arg Val Thr Arg Glu Ala Ala Gly 355 360 365 Pro Asn Ala Arg Leu
Tyr Ile Asn Asp Tyr Asn Thr His Glu Pro Lys 370 375 380 Lys Arg Asp
Phe Leu Tyr Asn Leu Val Arg Asp Leu Leu Ala Lys Gly 385 390 395 400
Val Pro Ile Asp Gly Val Gly His Gln Ser His Ile Arg Leu Glu Phe 405
410 415 Pro Ala Ile Asp Glu Met Glu Gln Ser Ile Glu Lys Phe Ala Ser
Leu 420 425 430 Gly Leu Asp Asn Gln Ile Thr Glu Leu Asp Met Gly Leu
Tyr Ser Asn 435 440 445 Asp Thr Asp His Tyr Glu Thr Ile Pro Glu Ala
Met Leu Ile Arg Gln 450 455 460 Ala His Arg Tyr Arg Ala Leu Phe Asp
Met Phe Ser Arg Gln Gln Glu 465 470 475 480 His Ile Ser Asn Val Thr
Ile Trp Gly Thr Asp Asp Gly Asn Thr Trp 485 490 495 Leu Ser Met Phe
Pro Ile Ala Arg Leu Asp Lys Pro Leu Leu Phe Asp 500 505 510 Glu Arg
Leu Lys Ala Lys Tyr Ala Tyr Trp Ala Leu Val Asp Pro Ser 515 520 525
Lys Val Pro Pro Leu Pro Ala Gly Ser Asn Glu 530 535
530DNAArtificial SequenceSLX-O9 sense primer 5ctcgagatgg atacattgaa
gttgtatgtg 30622DNAArtificial SequenceSLX-O9 antisense primer
6ggatccctat tcgttgctcc cc 22
* * * * *